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Human Cytomegalovirus Infection Upregulates the MitochondrialTranscription and Translation Machineries

S. Karniely,a* M. P. Weekes,b R. Antrobus,b J. Rorbach,c L. van Haute,c Y. Umrania,b D. L. Smith,d R. J. Stanton,e M. Minczuk,c

P. J. Lehner,b J. H. Sinclaira

Department of Medicine, University of Cambridge Clinical School, Addenbrookes Hospital, Cambridge, United Kingdoma; Cambridge Institute for Medical Research,University of Cambridge, Cambridge, United Kingdomb; MRC, Mitochondrial Biology Unit, Cambridge, United Kingdomc; Paterson Institute for Cancer Research, Universityof Manchester, Withington, Manchester, United Kingdomd; Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, United Kingdome

* Present address: Sharon Karniely, Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

ABSTRACT Infection with human cytomegalovirus (HCMV) profoundly affects cellular metabolism. Like in tumor cells,HCMV infection increases glycolysis, and glucose carbon is shifted from the mitochondrial tricarboxylic acid cycle to thebiosynthesis of fatty acids. However, unlike in many tumor cells, where aerobic glycolysis is accompanied by suppressionof mitochondrial oxidative phosphorylation, HCMV induces mitochondrial biogenesis and respiration. Here, we affinitypurified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changesupon HCMV infection. We found that the mitochondrial transcription and translation systems are induced early duringthe viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregu-lated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-inducedribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and sig-nificantly impaired viral growth under bioenergetically restricting conditions. Our findings demonstrate how HCMV ma-nipulates mitochondrial biogenesis to support its replication.

IMPORTANCE Human cytomegalovirus (HCMV), a betaherpesvirus, is a leading cause of morbidity and mortality during con-genital infection and among immunosuppressed individuals. HCMV infection significantly changes cellular metabolism. Akin totumor cells, in HCMV-infected cells, glycolysis is increased and glucose carbon is shifted from the tricarboxylic acid cycle to fattyacid biosynthesis. However, unlike in tumor cells, HCMV induces mitochondrial biogenesis even under aerobic glycolysis. Here,we have affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteomechanges upon HCMV infection. We find that the mitochondrial transcription and translation systems are induced early duringthe viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulatedby HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosomebiogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly im-paired viral growth. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication.

Received 12 January 2016 Accepted 25 February 2016 Published 29 March 2016

Citation Karniely S, Weekes MP, Antrobus R, Rorbach J, van Haute L, Umrania Y, Smith DL, Stanton RJ, Minczuk M, Lehner PJ, Sinclair JH. 2016. Human cytomegalovirus infectionupregulates the mitochondrial transcription and translation machineries. mBio 7(2):e00029-16. doi:10.1128/mBio.00029-16.

Editor Christine A. Biron, Brown University

Copyright © 2016 Karniely et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.

Address correspondence to Sharon Karniely, [email protected].

Human cytomegalovirus (HCMV) is a betaherpesvirus foundin 50% to 90% of human populations worldwide. Infection

of healthy individuals usually involves an asymptomatic acutephase followed by lifelong carriage in a latent form (1). However,HCMV is a serious pathogen under conditions of immunoincom-petence, being a leading cause of morbidity and mortality duringcongenital infection, bone marrow or solid organ transplanta-tions, or AIDS (1). HCMV drives major metabolic reprogram-ming of host cells during infection (2–5). Akin to tumor cells (6),HCMV infection leads to an increase in glucose uptake (7) byupregulation of glucose transporter 4 (Glut4) (8). During infec-tion, the flux of carbon through glycolysis is increased (2, 3), andglucose-derived citrate is shuttled from the mitochondria to thecytosol (cataplerosis) for the biosynthesis of fatty acids, vital for

viral envelopment (9). HCMV also drives an increase in glutamineconversion to �-ketoglutarate to fuel the tricarboxylic acid (TCA)cycle with carbon (anaplerosis) (10). While in some cancer cellsand in budding yeast the increase in glycolysis under aerobic con-ditions is associated with suppression of mitochondrial respira-tion and oxidative phosphorylation (OXPHOS), known as the“Crabtree effect” (11, 12), HCMV infection is associated with anincrease in both glycolysis (2) and mitochondrial respiration (13).Mitochondria play a central role in production of cellular energyand biosynthetic precursors and are key mediators and regulatorsof apoptosis and antiviral signaling (14). They contain autono-mous genomes that are expressed by unique transcription andtranslation systems, yet the human mitochondrial genome en-codes only 13 polypeptides (15). The vast majority of mitochon-

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drial proteins (700 to 1,000 in humans), including all of the pro-tein components of the mitochondrial transcription andtranslation machineries, are encoded by nuclear genes, are trans-lated in the cytosol, and are imported into mitochondria usingdedicated translocons (15).

HCMV infection is known to profoundly affect mitochon-dria and their function. Previous studies have indicated thatmitochondrial DNA (mtDNA) synthesis is stimulated byHCMV infection (16) and that upregulation of OXPHOS genesoccurs late during HCMV lytic cycle at the level of transcrip-tion (17), translation (18), and protein expression (19). Anincrease in endoplasmic reticulum-mitochondrial contact do-mains by HCMV infection has also been documented (20). Whilethe HCMV antiapoptotic protein UL37x1 (21) causes fragmenta-tion of the mitochondrial network (22), this is not associated withperturbed mitochondrial physiological functions and therefore isdifferent from other pathological conditions associated with net-work disassembly (23). On the contrary, HCMV-infected cells areknown to have increased labeling with the membrane potential-dependent dye tetraphenylphosphonium (TPP) (24) and showincreased oxygen consumption, indicative of induction of respi-ration (13). Viral UL37x1 may be involved in this process since aUL37x1 knockout virus was partially impaired in induction ofrespiration (13); HCMV �2.7 long noncoding RNA has also beensuggested to affect mitochondrial function by binding respiratorycomplex I (25). In this case, �2.7 RNA was required to sustain ATPproduction throughout viral infection (25).

How HCMV upregulates mitochondrial biogenesis and func-tion is not well understood. Here, we have used quantitative massspectrometry (MS) to measure the changes in the mitochondrialproteome following HCMV infection in order to understandthese processes. We found that multiple proteins of the mitochon-drial transcription and translation systems were induced earlyduring the viral replication cycle. This was accompanied by anincrease in the components of the respiratory chain complexesencoded by the mitochondrial genome. Proteins involved in mi-toribosome biogenesis were markedly induced by HCMV infec-tion. Knockdown of a recently identified member of this group,MRM3 (26, 27), abrogated virus induction of mitochondriallyencoded proteins and significantly impaired viral growth underbioenergetically restricting conditions.

RESULTSMapping HCMV-induced changes in the mitochondrial pro-teome using SILAC-MS. To better understand how HCMV stim-ulates mitochondrial biogenesis and functions, we mapped theHCMV-induced changes in the abundance of mitochondrial pro-teins using quantitative mass spectrometry (MS) based on stableisotope labeling by amino acids in cell culture (SILAC). We car-ried out a SILAC-based screen in U373 astrocytoma cells, which inour previous studies appear to be more sensitive to changes inmitochondrial function mediated by HCMV than fibroblasts (25)and gave higher yields and purity of mitochondria (unpublishedresults). Three parallel cultures of U373 cells were differentiallylabeled with SILAC media (containing “light,” “heavy,” or “me-dium” amino acids) and then infected with the HCMV Merlinstrain for 48 or 60 h or mock infected (see Fig. S1 in the supple-mental material). Following infection, the three populations ofcells were combined and mitochondria were isolated from thepooled-cell mixture by affinity purification. Lysed mitochondria

were digested into peptides followed by MS. We identified 1,171cellular proteins with at least 2 unique peptides in our mitochon-drial preparation (see Table S1A in the supplemental material).According to Gene Ontology (GO) annotation, 47% of identifiedproteins were mitochondrial, while 17% and 12% were annotatedto the closely associated organelles the endoplasmic reticulum(ER) and the nucleus, respectively (Fig. 1A). At 48 h postinfection(hpi), over 20% of identified proteins exhibited a specific andsignificant change using a 2-fold cutoff (Fig. 1B). Virus-inducedchanges to mitochondrial proteins persisted up to 60 hpi (seeFig. S2A in the supplemental material) and were reproducible (seeFig. S2B and Table S1B).

HCMV reduces the association of mitochondria with pro-teins of other cellular structures and downregulates antiviralproteins. To find functional groups within the proteins whoseabundance was significantly changed by HCMV infection, we

A

B

Mito47%

ER17%

Nuc12%

PM8%

Other16%

Subcellular distribu�on of iden�fied proteins

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1.E+09

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0.06 0.13 0.25 0.50 1.00 2.00 4.00 8.00 16.00

Intensity

H (mock)/L (48hpi)

FIG 1 SILAC-MS analysis of the changes in the mitochondrial proteomefollowing HCMV infection. (A) Subcellular localization annotation of 1,171human proteins identified in isolated mitochondria with at least 2 uniquepeptides represented as a percentage of the total. Gene Ontology (GO) infor-mation was imported using the UniProt database. (B) Scatter plot representingfold change in “heavy” (mock)/“light” (HCMV 48 hpi) protein abundance inisolated mitochondria. A total of 12.3% (144) of proteins were increased over2-fold following HCMV infection (red), 8.6% (101) were downregulated(blue), and 79.1% (926) were not significantly changed (gray).

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used the Database for Annotation, Visualization and IntegratedDiscovery (DAVID) (28). GO biological processes enrichedwithin downregulated proteins included the categories “Cell ad-hesion,” “Actin cytoskeleton organization,” “Glycolysis,” and“Response to virus” (see Table S2A in the supplemental material).Reduction in actin binding proteins is consistent with HCMVdisruption of the actin cytoskeleton (29–31). Given the docu-mented activation of glycolysis by HCMV (2, 3), the observedreduction in glycolytic enzymes may appear counterintuitive.However, we found that glycolytic enzymes were not downregu-lated during infection in total cell extracts (our unpublished data);rather, the association of these enzymes with mitochondria wasreduced in infected cells. Glycolytic enzymes are associated withmitochondria in mammalian cells (32). In plant cells (33, 34), theassociation of glycolytic enzymes with mitochondria is dynami-cally changed in response to respiratory demand (35). WhetherHCMV-induced disassociation of glycolytic proteins from mito-chondria has a metabolic role in HCMV replication remains to bedetermined. Of note, nonmitochondrial proteins were less associ-ated with mitochondria upon infection, likely due to virus-induced fragmentation of the mitochondrial network (22).

In U373 cells, we observed a downregulation of several antivi-ral proteins (see Table S2A in the supplemental material). Mostsignificant was an 8.3-fold reduction in the proapoptotic andproautophagic protein BCL-2/adenovirus E1B interacting protein3 (BNIP3). We also observed viral induction of acetyl-coenzyme Aacyltransferase 2 (ACAA2), which was shown to mitigate apopto-sis induced by BNIP3 (36). A reduction of the antiviral signalingproteins MAVS and STING, which act in interferon beta (IFN-�)induction through the IFN regulatory factor 3 (IRF3) pathway,was also seen. This reduction is in line with HCMV-induceddownregulation of IRF3 (19) and inhibition of IFN-� induction(37). A reduction in STING has also previously been observed intotal cell extracts of HCMV-infected fibroblasts (19), although nochanges in total amounts or subcellular localization of MAVSwere observed (19, 38). MAVS may therefore undergo differentfates in HCMV-infected fibroblasts and U373 cells. Our data pointto a reduction in two additional viral restriction factors implicatedin the interferon response: gamma-interferon-inducible protein16 (IFI16) and phospholipid scramblase 1. IFI16 is required foractivation of the IRF3 signaling cascade during human herpessimplex virus 1 (HSV1) infection (39). IFI16 was also shown torestrict HCMV replication, yet its inhibitory activity was not de-pendent on IFN-� induction (40). Phospholipid scramblase 1, arestriction factor of hepatitis B virus (41), was shown to augmentthe IFN response by increasing the expression of potent antiviralgenes (42).

Cellular pathways induced by HCMV infection. DAVIDanalysis of HCMV-upregulated proteins showed enrichment ofseveral cellular pathways, which included the KEGG_PATHWAYcategories “Arginine and proline metabolism,” “Fatty acid metab-olism,” and “One carbon pool by folate” (see Table S2B in thesupplemental material).

Within arginine metabolism-related proteins, the highest in-duction (8.7-fold) was found in arginase II (ARG2). L-Arginine isthe source for the synthesis of nitric oxide (NO), which plays amajor role in host defense against microbial infection. In mousemacrophages, induction of arginase II was shown to prevent NOproduction by depleting intracellular arginine pools (43). Induc-tion of arginase II was also previously detected during herpes sim-

plex virus 1 (HSV-1) infection of cornea (44). Consequently, itmay be that arginase II induction aids herpesviruses to suppress ahost NO antiviral response.

Fatty acid synthesis is induced by HCMV infection (9). In par-ticular, very-long-chain fatty acids are important for the produc-tion for infectious virions (45). HCMV-induced lipogenesis re-quires the inhibition of beta oxidation of long-chain fatty acidsand is mediated by recruitment of the cellular protein viperin tomitochondria (46, 47). Interestingly, we found that proteins in-volved in beta oxidation of short and medium fatty acids wereinduced by HCMV (see Table S2B in the supplemental material).These included the mitochondrial short-chain-specific acyl coen-zyme A (acyl-CoA) dehydrogenase (SCAD [2.3-fold induction])and medium-chain-specific acyl-CoA dehydrogenase (MCAD[4.4-fold induction]), which catalyze the first step of oxidation ofthese fatty acids. It also included mitochondrial acetyl-coenzymeA acyltransferase 2 (ACAA2 [3-fold induction]), which catalyzesthe last step of oxidation. In contrast, we found that the carnitine/acylcarnitine carrier protein, required for transport of long fattyacids into mitochondria, as well as the very-long-chain fatty acid-specific acyl-CoA dehydrogenase and trifunctional protein (TFP)complex, which catalyze beta oxidation of long-chain fatty acids,were not significantly changed by HCMV infection; this is consis-tent with the documented suppression of this pathway by HCMV(47). Whether a specific increase in the oxidation of short-chainfatty acids is supportive of HCMV replication remains to be de-termined. The induction of one-carbon metabolism in mitochon-dria is discussed below.

Mitochondria from HCMV-infected cells were enriched withproteins containing predicted N-terminal mitochondrial tar-geting sequences. By inspection of our list of mitochondrial pro-teins upregulated by infection, it appeared that mitochondrialmatrix proteins are much more increased than proteins of theother mitochondrial subcompartments. As most matrix proteinscontain N-terminal mitochondrial targeting peptides (mTP), weexpected an enrichment of proteins displaying a high probabilityof containing an mTP in our data set. We identified mTP scoresfor all of our quantified proteins using the TargetP 1.1 server (48).We then plotted the TargetP score against the log heavy/lightamino acid ratio (H/L ratio) (see Fig. S3A in the supplementalmaterial). Proteins with a negative log ratio were relatively en-riched in infected cells, which were labeled with light amino acids.We performed this analysis on data searched against the wholeUniProt database (see Fig. S3Aa) and against the UniProt databaseof mitochondrial proteins only (annotated by Mitocarta [49]) (seeFig. S3Ab). In both cases, the averaged log (ratio) for high TargetPscores was significantly lower than the averaged log (ratio) for lowscores, suggesting that proteins with high score of predicted mTPare indeed selectively enriched in HCMV-infected cells. Our find-ings may suggest that proteins containing an mTP are preferen-tially imported into mitochondria in infected cells. The reportedincreased accumulation of TPP in HCMV-infected cells (24) mayindicate an increase in the mitochondrial membrane potential ininfected cells, which is the driving force for import through theTIM complex into the matrix.

HCMV specifically upregulated proteins involved in expres-sion of the mitochondrial genome. Thirty-three of the 144 (23%)proteins upregulated by HCMV function in expression and main-tenance of the mitochondrial genome (Table 1). This group com-prised ~40% of virally upregulated proteins annotated “mito-

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TABLE 1 Fold change and suggested functions of mitochondrial expression factors upregulated by HCMV infection

Protein Gene(s)H/L ratio(mock/48 hpi)

No. of uniquepeptides Suggested mitochondrial function(s) Reference(s)

mTERF domain-containingprotein 1, mitochondrial

MREF3, MTERFD1 0.16 3 Mitochondrial ribosome biogenesis(mouse)

50

Mpv17-like protein 2 MPV17L2 0.19 2 Mitochondrial ribosome assembly(human)

51

Polymerase delta-interactingprotein 2

POLDIP2 0.22 6 Mitochondrial nucleoid associated(human)

100

Putative methyltransferaseNSUN4

NSUN4 0.25 3 Mitochondrial ribosome assembly(human, mouse)

52

28S ribosomal protein S27,mitochondrial

MRPS27 0.23 5 Small mitochondrial ribosomesubunit (bovine)

101

GTPase Era, mitochondrial ERAL1 0.25 7 Mitochondrial ribosome biogenesis(human)

53, 54

39S ribosomal protein L38,mitochondrial

MRPL38 0.26 7 Large mitochondrial ribosomesubunit (bovine)

102

39S ribosomal protein L37,mitochondrial

MRPL37 0.27 8 Large mitochondrial ribosomesubunit (bovine)

102

Dimethyladenosine transferase2, mitochondrial

TFB2M 0.28 5 Mitochondrial transcriptionactivation (mouse, human)

103



101



101

RNA methyltransferase-likeprotein 1

MRM3, RNMTL1 0.30 2 rRNA methyl transferase,mitochondrial ribosome biogenesis(mouse)

26, 27

GTP-binding protein 5 GTPBP5 0.31 3 Large mitochondrial ribosomeassociated, mitochondrialtranslation regulation (human)

104

Dimethyladenosine transferase1, mitochondrial

TFB1M 0.34 4 Mitochondrial transcription (human) 103

ATP-dependent RNA helicaseSUPV3L1, mitochondrial

SUPV3L1 0.35 9 DNA/RNA helicase, mitochondrialRNA turnover and processing(human)

105

Mitochondrial tRNA-specific2-thiouridylase 1

TRMU 0.35 4 Mitochondrial translation regulation(human)

106

Probable asparagine-tRNAligase, mitochondrial

NARS2 0.37 2 tRNA synthetase (human) 107

Mitochondrial ribonuclease Pprotein 1

TRMT10C, MRPP1 0.38 12 Mitochondrial tRNA maturation(human)

108

G-rich sequence factor 1 GRSF1 0.39 10 Mitochondrial mRNA turnover,mitochondrial ribosome biogenesis(human)

109

Putative ATP-dependent RNAhelicase DHX30

DHX30 0.40 23 Mitochondrial translation regulation(human)

110

Tyrosine—tRNA ligase,mitochondrial

YARS2 0.42 6 Mitochondrial tRNA synthetase(human)

111

Methionyl-tRNAformyltransferase,mitochondrial

MTFMT 0.44 2 Mitochondrial translation (human) 112


DAP3 0.44 11 Small mitochondrial ribosomesubunit (bovine)

101

tRNA modification GTPaseGTPBP3, mitochondrial

GTPBP3 0.44 4 Mitochondrial translation regulation(human)

113

SRA stem-loop-interactingRNA-binding protein,mitochondrial

SLIRP 0.45 3 Mitochondrial mRNA turnover(human)

114

Glycine—tRNA ligase GARS 0.46 6 Mitochondrial tRNA synthetase(human)

115



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chondrial” by GO, emphasizing the enrichment of this functionalgroup among mitochondrial proteins upregulated by infection.This group included proteins associated with the mitochondrialnucleoid, proteins involved in mitochondrial mRNA transcrip-tion and turnover, mitochondrial tRNA maturation enzymes, mi-tochondrial tRNA synthetases, components of the small and largesubunits of the mitoribosome, and proteins involved in mitoribo-some assembly (Table 1). We have confirmed that the measuredupregulation of mitochondrial expression proteins was not signif-icantly biased by normalization due to different total amounts ofheavy- and light-amino-acid-labeled mitochondrial proteins (seeFig. S3B and Text S1 in the supplemental material).

The most significant upregulation within this group was inproteins only recently identified to play a role in mitoribosomebiogenesis, a process which is still poorly understood. We ob-served a 6-fold viral induction of mTERF3 (50), a 5-fold induc-tion of MPV17L2 (51) and a 4-fold increase in NSUN4 (52),ERAL1 (53, 54) and MRM3 (RNMTTL1) (26, 27). Translationof mitochondrial proteins, like bacterial proteins, requires aninitiating formylated methionine. The formyl group is suppliedby one-carbon metabolism (55). Consistent with this, we alsoobserved an upregulation of enzymes that participate in one-carbon metabolism in the mitochondria, including mitochon-drial bifunctional methylenetetrahydrofolate dehydrogenase(MTHFD2 [5.2-fold]), monofunctional C1-tetrahydrofolatesynthase (MTHFD1L [2.2-fold]), and mitochondrial methionyl-tRNA formyltransferase (MTFMT [2.3-fold]).

Taken together, our SILAC data suggest that regulation ofmitochondrial translation is a key process in viral induction ofmitochondrial biogenesis. This is in line with a previous reportshowing that chloramphenicol (which specifically inhibits mi-tochondrial translation) abrogates HCMV induction of mito-chondrial respiration (13).

In contrast to the upregulation of mitochondrial genome ex-pression factors, the vast majority of nucleus-encoded proteins ofthe respiratory complexes were not affected by HCMV infection(see Table S3 in the supplemental material). Exceptions to thiswere several proteins suggested to be involved in assembly of therespiratory complexes, which involves the integration of nuclearlyand mitochondrially encoded proteins (highlighted in green inTable S3). These proteins included the complex I assembly factors,evolutionarily conserved signaling intermediate in Toll pathway(ECSIT), Mimitin (NDUFAF2) and NDUFAF7, and complex III

mitochondrial chaperone BCS1, as well as complex IV cyto-chrome c oxidase assembly factor 7.

Upregulation of mitochondrial expression factors occursearly during the HCMV replication cycle. We next used Westernblot (WB) analysis to confirm the changes detected in mitochon-drial expression factors in our SILAC-MS analysis and obtained agood correlation of measurements between methods (see Fig. S2Cin the supplemental material). Consistent with our SILAC-MSdata, we observed an upregulation of components of the small andlarge mitoribosomal subunits (MRPS 18b, with MRPL12 moresignificant for the former), mitochondrial ribosome biogenesisfactors (mTERF3 and MRM3), and mitochondrial transcriptionfactors (TFAM and TFB2M) in U373 cells at 48 hpi (Fig. 2, leftlanes). ATP5A a nuclearly encoded subunit of respiratory com-plex V, and the mitochondrial outer membrane protein porin(VDAC1) were not upregulated by HCMV infection of U373 cells,consistent with our SILAC data. We also analyzed if this upregu-lation occurs during HCMV infection of human fibroblasts,where induction of OXPHOS genes was previously shown (17,19), and found that upregulation of mitochondrial expressionproteins was more pronounced in HCMV-infected fibroblasts(Fig. 2, right lanes). In line with our SILAC-MS data, a minorupregulation of ATP5A and porin was observed in infected fibro-blasts, consistent with the documented overall increase in mito-chondrial biogenesis in these cells (13, 17, 19).

We performed a time course analysis to determine the kineticsof viral induction of mitochondrial expression factors usingMRM3 and TFB2M as representative proteins of the mitochon-drial translation and transcription machineries. In both U373 andfibroblast cells, the accumulation of MRM3 and TFB2M was evi-dent by 24 hpi, and they continued to accumulate through theviral replication cycle (Fig. 3A). Since upregulation was more ro-bust in fibroblasts, we continued our analysis in these cells.mtDNA copy number also increased during HCMV infection atlater stages of the viral replication cycle, in agreement with previ-ous reports (see Fig. S4 in the supplemental material) (13, 16).This increase could lead to an induction of mitochondrially en-coded transcripts (mRNAs and rRNAs). However, an increase inthe levels of mitochondrially encoded mRNAs per se may not besufficient (or even required) for increased synthesis of mitochon-drially encoded proteins as it was recently suggested that mamma-lian mitochondria (in vivo) contain a great excess of mitochon-drial transcripts (56). Of note, the 2.7-fold increase in mtDNA

TABLE 1 (Continued)

Protein Gene(s)H/L ratio(mock/48 hpi)

No. of uniquepeptides Suggested mitochondrial function(s) Reference(s)

Fast kinase domain-containingprotein 5

FASTKD5 0.47 6 Mitochondrial translation (human) 110

Single-stranded DNA-bindingprotein, mitochondrial

SSBP1 0.48 8 mtDNA replication 116

Elongation factor G,mitochondrial

GFM1 0.49 13 Mitochondrial translation (human) 117

Fast kinase domain-containingprotein 2

FASTKD2 0.49 9 Mitochondrial translation (human) 110

Zinc phosphodiesterase ELACprotein 2

ELAC2 0.49 9 Mitochondrial tRNA maturation(human)

118



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U373 HFFF2

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MRM3

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mTERF3

IE

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large ribosomal protein L12

TFB2M

TFAM

ATP5A

mt transcrip�on

Complex V

MOM

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0.00

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1.00

1.50

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2.50

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S18b L12 MRM3 mTERF3 TFB2M TFAM Porin ATP5A

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observed at 72 hpi is comparable to the ~3-fold increase inmtDNA synthesis (as measured by 3H-dT incorporation) at 68 hpiobserved by Furukawa et al. (16) and the 3.5-fold increase in mi-tochondrial mass (as assessed by nonyl acridine orange staining)observed by Kaarbø et al. (13). The ~300-fold increase in mtDNAat 72 hpi reported by Kaarbø et al. (13) was higher than our datasuggest.

Our results suggest that the upregulation of MRM3 andTFB2M involves early or possibly immediate early (IE) viral geneexpression. Consistent with this, no upregulation of MRM3 andTFB2M was observed when cells were infected with a UV-inactivated virus (which failed to produce the viral IE proteins)(Fig. 3B), and their induction was not perturbed by the viral DNApolymerase inhibitor phosphonoformic acid (PFA), which blocksviral late gene expression (marked by HCMV pp28) (Fig. 3B). Theinduction of MRM3 and TFB2M was also observed at the level ofmRNA (see Fig. S5 in the supplemental material), supporting theview that viral regulation of these genes is likely to be transcrip-tional.

HCMV genes known to target mitochondria are dispensablefor induction of mitochondrial expression factors. Two HCMVgene products have previously been shown to affect mitochon-drial respiration: the UL37x1 protein and the long noncoding�2.7 RNA. A UL37x1 knockout virus was partially impaired ininduction of respiration (13), while a �2.7 knockout virus failed tomaintain ATP production throughout the viral infection cycle(25). We therefore determined whether the UL37x1 and �2.7RNA genes are important for the induction of mitochondrial ex-pression factors observed at early stage of HCMV replication. Wefound that neither UL37x1 nor �2.7 is required for viral inductionof MRM3 and TFB2M (Fig. 4). The identity of the viral gene orgenes that govern this induction remains to be determined.

HCMV induces the production of mitochondrially encodedproteins. Having demonstrated that HCMV upregulates proteinsof the mitochondrial transcription and translation machineries,we asked whether mitochondrial translation and the end productsof these processes, mitochondrially encoded proteins, are also in-creased by HCMV infection. We first analyzed the assembly ofmitochondrial ribosomal proteins following infection using su-crose gradient separation. We found an increase in assembled mi-toribosomal subunits in the dense fractions using antibodiesagainst the large (MRPL12) (Fig. 5A) and small (MRPS18b[Fig. 5A] and MRPS17 [not shown]) ribosomal subunits.

We next looked at steady-state levels of mitochondrially en-coded proteins and found that mtDNA-encoded cytochrome oxi-dases 1 and 2 (mt-CO1 and mt-CO2), components of respiratorycomplex IV, were both upregulated after 48 h of infection of fi-broblasts (Fig. 5B; see Fig. S6A in the supplemental material). Incontrast, the levels of nuclearly encoded succinate dehydrogenaseA (SDHA), part of respiratory complex II, were not significantlychanged by infection. Of note, a moderate increase in mt-CO2 wasobserved in HCMV-infected U373 cells only at 72 hpi (seeFig. S6B) and not at 48 hpi (see Fig. S6B and Table S1A and S1B inthe supplemental material). The increased upregulation of mito-chondrially encoded proteins in infected fibroblasts compared to

U373 cells correlates with the robust upregulation of mitochon-drial transcription and translation factors in the former, as shownin Fig. 2.

We then followed mitochondrial translation by radiolabelingmock-infected and HCMV-infected cells with [35S]methionine inthe presence of emetine, which specifically blocks cytosolic (butnot mitochondrial) translation. When equal amounts of cell ly-sates were separated by SDS-PAGE and analyzed by autoradiog-raphy, we found that radiolabeling of mitochondrially encodedproteins was reduced after 24 h of HCMV infection (Fig. 5C, rightlanes; see Fig. S6C, lanes 1 and 2, in the supplemental material).This was surprising since our WB analysis (Fig. 5B) suggested anincrease in their steady-state levels. Moreover, we observed a sig-nificant decrease in the radiolabeling of cellular proteins (whenlabeling in the absence of emetine; Fig. 5C, left lanes). SinceHCMV, unlike other viruses, maintains cellular translation andmTOR activation (57), we questioned if the decrease in radiola-beled proteins we observed resulted from a specific reduction intranslation or reflected a reduction in [35S]Met uptake by infectedcells under our experimental conditions. We, therefore, measuredtrichloroacetic acid (TCA)-precipitable counts ([35S]Met incor-porated into proteins) and total radioactive count (including all[35S]Met taken up by the cells) in mock- and HCMV-infected celllysates. We found that, indeed, total [35S]Met counts in infectedcells (incorporated and nonincorporated [35S]Met) were reducedcompared to those in mock-infected cells, suggesting that less[35S]Met was taken up by infected cells. We then calculated cellu-lar and mitochondrial translation by dividing the TCA-precipitated counts by total counts, which represents the effi-ciency of [35S]Met incorporation into proteins and corrects for thebiases in uptake of [35S]Met into the cells. When the above wastaken into account, we found that mitochondrial translation isspecifically induced at 24 h post-HCMV infection, while rates ofcellular translation were similar between infected and uninfectedcells (Fig. 5D). Nonetheless, we cannot exclude the possibility thatHCMV perturbed cellular translation, which was accompanied bya decrease in uptake of [35S]Met, as translation inhibition waspreviously suggested to restrict uptake of at least some amino acids(58). In this case, our data would suggest a relative increase inmitochondrial translation over cytosolic translation in infectedcells but not an absolute increase in mitochondrial translationcompared to that in uninfected cells.

Interestingly, when uninfected cells were pretreated for 4 hwith emetine prior to radiolabeling, mitochondrial translationwas essentially blocked, while translation in HCMV-infected cellswas maintained (see Fig. S6C in the supplemental material). It waspreviously shown that inhibition of cytosolic translation blocksmitochondrial translation within several hours, probably due toan elimination of a short-lived nucleus-encoded mitochondrialtranslation factor or factors (59). In sucrose gradients, we haveobserved an increase not only in assembled ribosomal subunitsbut also in nonassembled MRPL12 (Fig. 5A, fractions 1 to 4).Having a pool of mitochondrial translation factors (includingnonassembled ribosomal subunits) may allow infected cells to re-

FIG 2 Upregulation of mitochondrial translation and transcription factors in HCMV-infected cells. U373 and human fibroblast (HFFF2) cells were either mockinfected or infected with HCMV (Merlin) at an MOI of 5 for 48 h. WB analysis of total cell extracts using the indicated antibodies is shown. The fold change inprotein abundance measured by WB is presented in the column charts.




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tain mitochondrial translation under stress that blocks cytosolictranslation.

To determine if the stability of mt-CO1 and mt-CO2 was alsoaffected by viral infection, we blocked mitochondrial translation at

24 hpi using chloramphenicol and monitored the mt-CO1 and mt-CO2 levels at several time points. We found that the stability of bothmt-CO1 and mt-CO2 was increased by infection (see Fig. S6D in thesupplemental material). Blocking either mitochondrial transcription,

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FIG 3 Upregulation of mitochondrial expression factors occurs during the early phase of HCMV replication. (A) Time course analysis. U373 and HFFF2 cells wereeither mock infected or infected with HCMV (Merlin) at an MOI of 5 for the times indicated. Lysates were processed and analyzed as described in the legend to Fig. 2.Membranes were labeled with the indicated antibodies. The upregulation of mitochondrial expression factors was first observed at 24 hpi concomitant with theexpression of the HCMV UL44 early protein. m., mock infected cells. (B) Induction of mitochondrial expression factors is dependent on the expression of immediateearly/early but not late HCMV genes. HFFF2 cells were either mock infected or infected with an intact or a UV-inactivated HCMV (Merlin) at an MOI of 5 for 48 h.

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using ethidium bromide (EtBr) (Fig. 5B), or mitochondrial transla-tion (see Fig. S6A) immediately after viral inoculation caused, as ex-pected, an almost complete elimination of mt-CO1 and mt-CO2 butnot of nucleus-encoded SDHA from both infected and uninfectedcells.

This inhibition of mitochondrial transcription and translationdid not feed back to the nucleus to block expression of nucleus-encoded MRM3 and TFB2M (which are required for these pro-cesses); viral induction of both proteins was not impaired by EtBror chloramphenicol treatments (Fig. 5B; see Fig. S6A in the sup-plemental material).

Inhibition of mitochondrial ribosome biogenesis interfereswith viral growth. Why might HCMV require an increase in mi-tochondrial gene expression? Blocking mitochondrial transcrip-tion or translation after viral infection did not affect the accumu-lation of immediate early or late viral proteins at 48 hpi, suggestingthat expression of the mitochondrial genome is not required forviral gene expression (Fig. 5B; see Fig. S6A in the supplementalmaterial). To evaluate the importance of mitochondrial transla-tion for viral growth, we blocked mitochondrial translation after24 h of infection (the earliest time point where we observed aninduction in mitochondrial translation factors) and measured re-leased virus titers at 120 hpi. Chloramphenicol treatment caused astatistically significant, yet very minor, ~2-fold reduction in virustiters when cells were grown on Dulbecco’s modified Eagle’s me-dium (DMEM) supplemented with glucose (5 mM) (Fig. 6A,compare the 1st column pair), a similar effect was reportedpreviously (13). While not leading to cell death (not shown),this mild effect may result from a slightly poorer health ofchloramphenicol-treated cells. No significant effect on titers wasseen earlier during infection (at 3 days postinfection [dpi]), yet

treatment of cells with chloramphenicol 24 h prior to infectioncaused a �0.5-log reduction in viral titers (see Fig. S6E in thesupplemental material). Importantly, we observed a robust �1.5log reduction of viral growth when mitochondrial translation wasblocked under more restrictive conditions. For instance, cellgrowth on galactose as a major carbon source is more dependenton oxidative phosphorylation and is thus routinely used to testrespiratory competence of cells (60). When HCMV-infected cellswere shifted to growth on galactose at 24 hpi, only a minor reduc-tion in virus titers at 120 hpi was observed compared to growth onmedia containing glucose (Fig. 6A, compare black columns 1 and3). However, a significant �1.5-log reduction in virus titers oc-curred when chloramphenicol was added to galactose-fed infectedcells (Fig. 6A, compare the 3rd column pair), suggesting an im-portant role for mitochondrial translation under demanding re-spiratory conditions. No significant death of infected cells wasassociated with growth on galactose either with or without chlor-amphenicol (data not shown). Impaired growth of cultured cellstreated with chloramphenicol is driven by pyrimidine auxotrophydue to the requirement of a mitochondrial electron transportchain for the activity of dihydroorotate dehydrogenase (DHOde-hase), a key enzyme in pyrimidine biosynthesis (61). Accordingly,supplementation of chloramphenicol-treated cells with uridinerestores their growth to normal levels (61). Since pyrimidine bio-synthesis is important for HCMV replication (62), and ourSILAC-MS data show HCMV induction of DHOdehase (see Ta-ble S1A in the supplemental material), we asked if supplementa-tion of HCMV-infected cells with uridine would augment viralgrowth when mitochondrial translation was inhibited. However,uridine addition alone could not alleviate the inhibition of viralproduction on cells grown on galactose in the presence of chlor-

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FIG 4 HCMV �2.7 and UL37x1 are not required for the induction of mitochondrial expression factors. HFFF2 cells were either mock infected or were infectedfor 48 h with wild-type (WT) HCMV (Merlin-BAC in panel A or Towne-BAC in panel B) or mutant viruses with the �2.7 (A) or UL37x1 (B) genes deleted.




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amphenicol (Fig. 6A, compare 4th column pair). Thus, the re-quirement of mitochondrial translation is not due to impairmentof pyrimidine production. This fits our observation that chloram-phenicol does not prevent late viral protein production (Fig. 5B),

as would be expected if pyrimidine biosynthesis was impaired.Taken together, our findings suggest that chloramphenicol inhib-its viral replication due to an energetic deficiency.

Our SILAC-MS data demonstrated that proteins involved in

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FIG 5 HCMV induces the synthesis and stabilization of mitochondrially encoded proteins. (A) Assembled mitoribosomes are induced by HCMV. Equalamounts of proteins of total cell lysates from mock-infected or HCMV-infected HFFF2 cells at 48 hpi were separated on a linear sucrose gradient (10 to 30%[wt/vol]) and analyzed by WB. Free nonassembled ribosomal subunits (SSU, small subunit; LSU, large subunit) migrate to the lower fractions, while assembledmitoribosomes appear in the higher-density fractions. (B) Induction of mitochondrially encoded proteins by HCMV. HFFF2 cells were either mock infected orwere infected with HCMV at an MOI of 5. After 2 h, the inoculum was washed, and cells were refreshed with untreated medium (UT) or with medium containingethidium bromide (EtBr) to block mitochondrial transcription. Cells were harvested at 48 hpi, and lysates were processed and analyzed as described in the legendto Fig. 2. The chart shows the fold change in protein abundance. HCMV induces mitochondrial translation. (C to D) Mock-infected and HCMV-infected cellswere radiolabeled with [35S]methionine at 24 hpi in the presence of emetine (which blocks cytosolic translation) to determine mitochondrial translation or in theabsence of emetine to determine cellular translation. Total cell lysates were separated on a 4 to 12% Bis-Tris Plus PAGE. Equal loading of the gels was confirmedby staining the gel with Coomassie brilliant blue G-250. Dried gels were exposed to a phosphorimager screen and visualized using a phosphorimager scanner (C).Radioactive counts in cell lysates were measured directly or after TCA precipitation and used to calculate cellular and mitochondrial translation efficiencies, asdescribed in the text. The chart shows the fold change in translation after infection (D).

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mitochondrial ribosome biogenesis are highly induced uponHCMV infection. On this basis, we asked if depletion of such avirally induced protein could prevent the induction of mitochon-drial translation and impair viral growth. Consequently, we trans-fected fibroblasts with either a control small interfering RNA(siRNA) or siRNA directed against the virus-induced mitoribo-somal biogenesis factor MRM3. After 16 h, cells were infected withHCMV and shifted to galactose medium at 24 hpi. At 120 hpi, wemeasured viral titers and analyzed cells by WB analysis. We foundthat depletion of MRM3 (~20% relative to control siRNA) abro-gated viral induction of the mitochondrially encoded CO2 (mt-CO2). In contrast, nucleus-encoded OXPHOS proteins were notsignificantly affected by MRM3 depletion (Fig. 6B). Concomi-tantly, knockdown of MRM3 significantly reduced viral titers(Fig. 6C), which mimicked the effect of chloramphenicol(Fig. 6A). Thus, viral induction of this mitoribosomal assemblyfactor appears important for viral growth.

DISCUSSION

HCMV has a profound effect on the expression of mitochondrialgenes as well as on mitochondrial organization and functions.Here, we have affinity purified mitochondria and used quantita-tive MS to map the HCMV-mediated changes in the mitochon-drial proteome. We found that in HCMV-infected U373 cells,over 20% of mitochondrially associated proteins were signifi-cantly modulated. We identified several antiviral factors that weredownregulated by HCMV infection, which included proteins in-volved in regulation of the interferon response, a major target ofHCMV (37). Interestingly, we found that a large group of mito-chondrial proteins—all involved in mitochondrial gene expres-sion—were significantly upregulated during infection. This up-regulation was more robust in infected fibroblasts than in U373cells, which is consistent with the more efficient viral replication infibroblasts (63). The highest fold change occurred in proteins in-volved in the biogenesis of the mitoribosome. Although, the struc-ture of the mitoribosome was recently resolved at high resolution(64), the process of mitoribosome biogenesis is still largely un-known, and most mitochondrial biogenesis factors we found to bevirally induced have only recently been functionally annotated. Itis tempting to speculate that additional mitochondrial proteinsupregulated by HCMV infection that currently have no annota-tion might have a role in mitoribosome biogenesis.

HCMV induction of proteins involved in mitochondrial tran-scription and translation was correlated with an increase in[35S]methionine incorporation into mitochondrial proteins fol-lowed by an increase in steady-state levels of mitochondrially en-coded proteins (Fig. 5). Interestingly, mitochondrially encodedproteins were induced by HCMV infection at times when geneexpression of nucleus-encoded mitochondrial proteins was un-changed. Thus, the upregulation in the mitochondrial transcrip-

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FIG 6 Inhibition of mitochondrial translation reduces virus titers. (A)HFFF2 cells were infected with HCMV at an MOI of 5 for 1 h and then washedand refreshed with DMEM supplemented with 10% dialyzed FBS, 2 mM glu-tamine, and antibiotics (DMEM-10dFBS) and 5 mM glucose. At 24 hpi, cellswere washed and refreshed with DMEM-10dFBS containing either 5 mM glu-cose or 5 mM galactose with or without the addition of 0.2 mM uridine. Eachmedium was either left nontreated (nt) or was treated with chloramphenicol(50 �g/ml) to block mitochondrial translation. At 5 dpi, media were collectedfrom cells, and released virus titers in supernatants were quantified using the50% tissue culture infective dose (TCID50). Error bars represent the standarderror of the mean (SEM) from two experiments with three replications each.(B and C) HFFF2 cells were transfected with a control siRNA or an siRNAtargeting MRM3. After 22 h of transfection, cells were infected with HCMV

(Continued)

Figure Legend Continued

(Merlin) at an MOI of 3, and at 24 hpi, cells were washed and refreshed withDMEM-10dFBS containing 5 mM galactose. At 5 dpi, media were collectedfrom infected cells, and virus titers were quantified using the TCID50 (C). Cellswere lysed and analyzed by WB; the results of two biological repeats are shown(B). The chart shows the fold change in protein abundance compared to con-trol siRNA-treated cells. Error bars in panel C represent the standard errorsfrom two biological repeats, each performed with three transfection replicates.*, P � 0.05; **, P � 0.001 (unpaired t test with Welch’s correction).




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tion and translation apparatuses may prime the biogenesis of themitochondrial respiratory complexes. Several lines of biochemicalevidence suggest that the mitochondrially encoded subunits mayform a scaffold during the assembly of respiratory chain com-plexes that is then joined by nucleus-encoded subunits importedfrom the cytosol (65, 66).

Unlike the early induction of mitochondrial genome expres-sion factors, the increase in mitochondrial DNA copy number(13) (see Fig. S4 in the supplemental material) and in mitochon-drial mass (13) occurs only at late stages of HCMV replication. Asimilar sequence of events occurs during Arabidopsis germination(67). In dry plant seeds, mitochondria appear in typical metabol-ically inert structures, termed promitochondria, and undergorapid development and multiplication into mature metabolicallyactive mitochondria following imbibition (the passive uptake ofwater by the seed) (68, 69). A detailed transcriptional analysisthroughout the germination of Arabidopsis thaliana seeds revealedthat transcripts encoding mtRNA transcription, editing and splic-ing factors peak early during germination. These are closely fol-lowed by transcripts of mitochondrial OXHPOS genes and bymitochondrial translation factors. The expression of nuclearOXHPOS genes lagged behind that of their mitochondrial coun-terparts (67). It may be that the sequence of events observed dur-ing the induction of mitochondrial biogenesis in HCMV-infectedcells and in germinating Arabidopsis seeds represents an evolu-tionarily conserved process. Importantly, while mitochondrialtranslation precedes the increase in mitochondrial mass and mi-tochondrial DNA copy number during HCMV replication, it ap-pears not to be essential for their induction as neither was blockedby chloramphenicol (13).

What controls mitochondrial biogenesis during HCMV repli-cation? Our data strongly suggest that this is a virally driven pro-cess, as it requires active expression of the viral genome and fails tooccur using a UV-inactivated virus. We show that mitochondrialexpression factors start to accumulate at 24 hpi, suggesting thatviral early genes are involved, although the involvement of IE geneexpression cannot be excluded. However, neither UL37x1, an IEprotein that localizes to mitochondria with early kinetics (21, 22,70, 71), nor the early �2.7 noncoding RNA (25) (both with doc-umented effects on mitochondrial function) was required for theinduction of MRM3 and TFB2M (Fig. 4). The identities of theviral genes that govern this induction remain to be found.

The coordinated induction of mitochondrial transcription andtranslation factors is likely to be mediated by a cellular regulator,possibly activated by a viral gene product. Seo et al. have previ-ously shown that at 24 h of HCMV infection, viral UL37x1 recruitsthe cellular protein viperin to mitochondria, where it interfereswith fatty acid beta oxidation, causing a 50% reduction in cellularATP (46). At 72 hpi, however, viperin was relocalized from mito-chondria to the cytoplasmic viral assembly compartment (46).This may explain why we have not identified viperin by MS in ourmitochondrial preparations at 48 or 60 hpi. We show that theinduction of mitochondrial expression factors initiates at 24 hpi.While this induction was independent of UL37x1 (Fig. 4), it re-mains possible that mitochondrial biogenesis is induced to com-pensate for an initial perturbation of ATP synthesis which thenallows ATP levels to be maintained later during HCMV infection.We have previously reported that following an initial (more sub-tle) reduction in cellular ATP following HCMV infection of U373cells, ATP levels persist for at least 5 days postinfection (25). A

compensatory increase in mitochondrial biogenesis was reportedfor multiple respiratory chain dysfunctions (72–74) and was pro-posed to explain an increase in mitochondrial mass in certaintypes of tumors (75).

Although several master regulators of mitochondrial biogene-sis have been identified (reviewed in reference 76), to our knowl-edge, there is no transcription factor yet known to specificallyregulate the genes encoding mitochondrial transcription andtranslation proteins. Peroxisome proliferator-activated receptorgamma coactivator 1-alpha (PGC-1�) is a transcriptional coacti-vator that is induced under physiologic conditions that requiremitochondria to produce heat or ATP (76). Although PGC-1�transcription is also induced during HCMV infection of fibro-blasts (13), the late kinetics of induction of PGC1� by HCMVsuggests that PGC-1� is not the initiator of induction of mito-chondrial genome expression we observed during infection. How-ever, PGC-1� may be involved in the late increase in mitochon-drial mass. Nuclear respiratory factors 1 and 2 (Nrf1 and 2) aretranscription factors that regulate the expression of nucleus-encoded respiratory complex subunits and genes involved inmtDNA transcription and replication, heme biosynthesis, and mi-tochondrial protein import (76). Nrf2, which also regulates anti-oxidant genes (77), is induced early during HCMV infection andprotects infected cells from oxidative stress (78). Importantly,knockdown of Nrf2 does not cause any inhibition of HCMVgrowth under nonstressed conditions (78), unlike the reductionobserved upon mitochondrial translation block (13) (Fig. 6).Nonetheless, involvement of Nrf2 in HCMV induction of mito-chondrial expression factors cannot formally be excluded. Somegenes involved in mitochondrial biogenesis are known to be reg-ulated at the level of translation. mTORC inhibitors suppress thetranslation of mRNAs encoding components of respiratory com-plex V (ATP synthase), complex I assembly factors, as well as themajor mitochondrial transcription factor TFAM and several mi-toribosomal proteins (79). Analysis of total and polysome-associated mRNAs from HCMV-infected cells suggested that thetranslation of several mitoribosomal subunits was induced by thevirus (18). Some OXPHOS and mitoribosomal proteins weretranslationally induced in noninfected cells expressing the HCMVmTORC1 activator UL38, while others, induced by the virus, werenot induced by UL38 alone. Our analysis suggests that, at least forMRM3 and TFB2M mitochondrial expression factors (see Fig. S5in the supplemental material), regulation occurs at the level oftranscription.

Our analysis showed that mitochondrial translation is impor-tant for HCMV replication, especially under conditions requiringactive mitochondrial respiration. Specific inhibition of mitochon-drial translation by chloramphenicol or by knockdown of the mi-toribosome biogenesis factor MRM3 caused an ~1.5-log reduc-tion in virus titers in galactose-fed cells (Fig. 6A). This inhibitionof viral growth by chloramphenicol could not be rescued by theaddition of uridine (Fig. 6A), previously shown to recover cellgrowth arrest by chloramphenicol (61). Taken together, thesefindings suggest that mitochondrial translation is important tokeep mitochondria bioenergetically active during viral replica-tion.

How do other viruses affect mitochondrial biogenesis? Rubellavirus infection causes a significant increase in the activity of mito-chondrial respiratory complexes II and III, as well as an increase inmitochondrial membrane potential and cellular ATP levels (80,

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81). Increases in mitochondrial membrane potential and ATP alsooccur during persistent infection with measles virus (82). Similarto HCMV, these viruses also have protracted life cycles that mayrequire prolonged maintenance of mitochondrial activity. Ofnote, among their many functions, interferons have been shownto inhibit mitochondrial gene expression (83–86), supporting thenotion that mitochondrial function is supportive of viral replica-tion. In contrast, many viruses with a short replication cycle inter-fere with mitochondrial activity (87). This is well demonstratedfor HSV-1 and HSV-2, which unlike HCMV, have a much shorterreplication cycle, that in culture ends with abrupt cytolysis of in-fected cells. Both HSV-1 infection and HSV-2 infection result inreduced levels of cellular ATP and mitochondrial membrane po-tential at late stages of HEp-2 infection (88). Similarly, the HSV-1US3 protein is known to induce inhibition of mitochondrial res-piration in HeLa cells (89). Moreover, in contrast to the inductionof mtDNA synthesis by HCMV, HSV-1 encodes a mitochondri-ally targeted nuclease, UL12.5, which degrades mtDNA early dur-ing HSV-1 infection (90). Although the importance of mtDNAelimination for HSV-1 replication is not understood (90), it isclear that HSV-1 does not require active and proliferating mito-chondria, apparently quite unlike HCMV. The importance of mi-tochondrial integrity as a cue for an immune response has recentlybecome clear. mtDNA can serve as a stress signal both within andoutside the cell. mtDNA released from cells induces inflammationby activation of polymorphonuclear neutrophils (PMNs) throughCpG-Toll-like receptor 9 (TLR9) interactions (91). Within cells,mtDNA released from mitochondria during apoptosis is sensedby the cytosolic cGAS DNA sensor and leads to the induction oftype I interferons (92–94). It will be interesting to determinewhether, in addition to its metabolic and bioenergetic functions,induction of mitochondrial biogenesis is important for keepingHCMV-infected cells immunologically silent.

MATERIALS AND METHODSCells and viruses. Primary human fetal foreskin fibroblast cells (HFFF2)and U373 cells were grown in Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 10% (vol/vol) heat-inactivated fetal bovineserum (FBS), 100 �g/ml penicillin-streptomycin, and 2 mM L-glutamineat 37°C in 5% CO2. The HCMV strain Merlin (95) was used in all exper-iments, except if stated otherwise. The Towne-BAC (bacterial artificialchromosome) �UL37x1 mutant HCMV and its parental strain (96) werea kind gift from E. Mocarski (Emory University School of Medicine, At-lanta, GA). The Merlin BAC-derived clone was used for the constructionof a ��2.7 strain by recombineering (see Text S1 in the supplementalmaterial). For the SILAC experiment, media collected from HCMV(Merlin)-infected cells were spun at 13,530 g (average) for 2 h at 4°C topellet viruses. Pellets were then resuspended in SILAC Minimum Essen-tial Medium (lacking lysine and arginine) supplemented with 10%dialyzed FBS.

Virus infection. Cells seeded 1 day prior to infection were inoculatedwith HCMV at a multiplicity of infection (MOI) of 5 for 2 h unless indi-cated differently and washed once in medium. Where indicated, cells wereincubated with PFA at 300 �g/ml, chloramphenicol at 50 or 100 �g/ml,and EtBr at 40 or 80 ng/ml from the time of infection onwards. For infec-tion of the �UL37x1 mutant and its parental strain (where viral titers werelow), we applied a spin infection protocol, in which plated cells were spunwith virus for 30 min at 730 g at room temperature followed by 1 h ofincubation in a humidified CO2 incubator.

SILAC. U373 cells were cultured in SILAC DMEM supplemented with10% dialyzed FBS (dFBS), 100 �g/ml penicillin-streptomycin, and 2 mML-glutamine. SILAC medium was supplemented with either “light” (Arg 0,

Lys 0 [Sigma]), “medium” (Arg 6, Lys 4, [Cambridge Isotope Laborato-ries]), or “heavy” (Arg 10, Lys 8 [Cambridge Isotope Laboratories])amino acids at 50 mg/liter and L-proline at 280 mg/liter. Incorporation of“heavy” label was �98% for both arginine and lysine-containing peptides.Cells were propagated for 7 doublings in SILAC medium to enable incor-poration of the labeled amino acids into cellular proteins. Twenty-fourhours prior to infection, 75-cm2 flasks were seeded with 3.2 � 106 cells.Two flasks were established for each SILAC medium (“light,” “medium,”and “heavy”). Cells were sequentially infected at an MOI of 3 with HCMVstrain Merlin (prepared in SILAC DMEM). Mock-infected cells (“heavy”)were inoculated with conditioned medium from HFFF2 cultured cells.Forty-eight-hour (“light”) and 60-h (“medium”) infections were stag-gered such that all flasks were harvested simultaneously. Infection effi-ciency was monitored using HCMV IE1 immunostaining.

Mitochondrial isolation. Mitochondria were affinity purified usingsuperparamagnetic microbeads conjugated to anti-TOM22 antibodies(Miltenyi Biotec) (see Text S1 in the supplemental material).

Mass spectrometry and data analysis. Mass spectrometric analysis,database searching, and data processing were performed as describedpreviously (97). Briefly, for the three-label experiment (Fig. 1; seeTable S1A in the supplemental material), mitochondrial protein ex-tracts were separated by gel electrophoresis and divided into 24 gelslices followed by in-gel trypsin digestion. Liquid chromatography-tandem MS (LC-MS/MS) was performed using a NanoAcquity ultra-performance liquid chromatograph (uPLC) (Waters, Milford, MA)coupled to an LTQ-OrbiTrap XL (Thermo Scientific, Tampa, FL).Peptides were eluted using a gradient rising to 25% MeCN by 70 minand 45% MeCN by 80 min. MS data were acquired between 300 and2,000 m/z at 60,000 full width at half-maximum (fwhm). Collision-induced dissociation (CID) spectra were acquired in the LTQ devicewith MS/MS switching operating in a top-6 data-dependent acquisi-tion (DDA) fashion triggered at 500 counts.

For the two-label biological repeat (see Fig. S2B and Table S1B in thesupplemental material), mitochondrial protein extracts were dialyzed andtrypsinized on a column as described previously (97). Eluted peptideswere fractionated off-line by high-pH reverse-phase high-performanceliquid chromatography (HpRP-HPLC) using a Dionex UltiMate 3000powered by an ICS-3000 SP pump with an Agilent Zorbax Extend-C18

column (4.6 mm by 250 mm, 5-�m particle size). Mobile phases (H2Oplus 0.1% NH4OH or MeCN plus 0.1% NH4OH) were adjusted topH 10.5 with the addition of formic acid, and peptides were resolved usinga linear 40-min 0.1% to 40% MeCN gradient over 40 min at a flow rate of400 �l/min and a column temperature of 15°C. Eluting peptides werecollected in 15-s fractions. LC-MS/MS was performed on 42 combinedfractions as described above but with a gradient rising to 45% MeCN by100 min and MS/MS triggered at 1,000 counts.

Raw data files were processed as previously described (97) using Max-Quant version 1.3.0.5 (98) and a merged Homo sapiens/HCMV strain MerlinUniProt database (downloaded 9/5/14). Gene ontology cellular compart-ment (GOCC) terms were added using the UniProt database (99).

Pathway analysis. The Database for Annotation, Visualization andIntegrated Discovery (DAVID) was used to determine Gene Ontologybiological processes (GOTERM_BP) and KEGG pathway enrichment(28). The groups of virus-induced downregulated and upregulated pro-teins were searched against a background of all proteins quantified withinthe relevant experiment.

Analysis of the mitochondrial ribosome profile on density gradi-ents. Total cell lysates in (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100,supplemented with protease inhibitor cocktail [Roche]) were loaded on alinear sucrose gradient (2 ml 10 to 30% [wt/vol]) in a mixture of 50 mMTris-HCl (pH 7.2), 10 mM Mg(OAc)2, 40 mM NH4Cl, and 25 mM KCl andcentrifuged for 2 h 15 min at 100,000 � gmax at 4°C (39,000 rpm [BeckmanCoulter TLS-55 rotor]). Twenty fractions (100 �l each) were collected, and10-�l aliquots of the first 17 fractions containing mitoribosomal subunits andassembled mitoribosomes were analyzed by WB.




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Mitochondrial translation analysis. Mock-infected and HCMV-infected cells were incubated for 30 min in L-methionine-free DMEM sup-plemented with 10% dialyzed fetal bovine serum (dFBS) and emetine(100 �g/ml) to block cytosolic translation and 250 �Ci/ml [35S]methionine(EasyTag L-[35S]methionine [PerkinElmer, Waltham, MA]). For total cellu-lar translation, 25 �Ci/ml was used without emetine. After labeling, cells werewashed twice with excess phosphate-buffered saline (PBS) before lysis withcold radioimmunoprecipitation assay (RIPA) buffer supplemented withcomplete protease inhibitor cocktail (Sigma-Aldrich). Insoluble material wasremoved from the lysate by centrifugation for 10 min at 10,000 � g at 4�C.Equal amounts of protein were spotted onto Whatman filter papers in 4replicates. After drying, two replicates were used to measure the total countsper minute ([35S]Met incorporated into proteins � [35S]Met taken up by thecells yet not incorporated), while two replicates were TCA precipitated tomeasure the acid-insoluble portion ([35S]Met incorporated into proteinsonly) by incubation of 20 min in cold 10% TCA, boiling for 15 min in 5%TCA, and washing in 5% TCA followed by washing in 95% ethanol anddrying. Radioactive counts were measured after addition of Ultima Gold scin-tillation liquid (PerkinElmer) using a Tri-Carb 1500 liquid scintillation ana-lyzer (PerkinElmer). Translation was calculated as TCA-precipitated counts/total counts. For autoradiography analysis, lysates were separated on 4 to 12%Bis-Tris Plus PAGE, after which the gel was stained with Coomassie brilliantblue G-250 (to confirm equal loading), dried, exposed to a phosphorimagerscreen, and visualized using a FLA5100 scanner (Fujiimager, Tilburg, TheNetherlands).

Analysis of the stability of mitochondrially encoded proteins.Mock-infected or HCMV-infected cells at 24 hpi were treated with chlor-amphenicol (50 �g/ml) to block mitochondrial translation. Cell extractswere made after 0, 6, 12, 24, and 30 h of addition of chloramphenicol andanalyzed by WB for mt-CO2, mt-CO1, and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) as a loading control.

Effect of chloramphenicol treatment on viral titers. Cells were in-fected with HCMV (Merlin) at an MOI of 5 for 1 h, washed once, andrefreshed with DMEM supplemented with 10% dFBS, penicillin/strep-tomycin, and 2 mM L-glutamine (DMEM-10dFBS) and 5 mM glucose.At 24 hpi, cells were washed and refreshed with DMEM-10dFBS me-dium containing either 5 mM glucose or 5 mM galactose (galactose-DMEM-10dFBS) with or without the addition of 0.2 mM uridine. Eachmedium was either left untreated or treated with chloramphenicol(50 �g/ml) to block mitochondrial translation. At 5 dpi, media werecollected from cells and kept at �80°C until assayed for determinationof viral titers (see Text S1 in the supplemental material). Cells weretested for propidium iodide exclusion to determine viability.

siRNA knockdown of MRM3. RNA interference (RNAi) duplexes(see Text S1 in the supplemental material) were delivered to 105 cells in12-well culture plates 1 day prior to infection with HCMV using oligo-fectamine transfection reagent (Life Technologies). Infection was carriedout at an MOI of 3 in penicillin-streptomycin-free DMEM-glucose. After24 h of infection, cells were shifted to galactose-DMEM-10dFBS. After5 days of infection, media were collected from cells and spun at 800 g for10 min at 18°C. Supernatants were kept frozen at �80°C until assayed forviral titers. The experiment included three transfection replicates and twobiological repeats. Of the three replicate wells, one well was harvested forWB analysis, while the remaining two wells were used to determine cellviability by trypan blue exclusion using a Countess automated cell counter(Life Technologies).

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at http://mbio.asm.org/lookup/suppl/doi:10.1128/mBio.00029-16/-/DCSupplemental.

Text S1, DOCX file, 0.03 MB.Figure S1, PDF file, 0.2 MB.Figure S2, PDF file, 1.3 MB.Figure S3, PDF file, 1.2 MB.Figure S4, PDF file, 0.1 MB.Figure S5, PDF file, 0.1 MB.

Figure S6, PDF file, 1.7 MB.Table S1, XLSX file, 0.3 MB.Table S2, XLSX file, 0.01 MB.Table S3, XLSX file, 0.02 MB.

ACKNOWLEDGMENTS

We are grateful to Linda Teague and Roy Whiston for excellent tech-nical assistance. We thank Sigal Ben-Yehuda, Einav Gross, OphryPines, Ann Saada, Antonella Spinazzola, Noam Stern-Ginossar, AlbertTaraboulos, and Dana Wolf for useful discussions and assistance withreagents.

S.K. was supported by a European Molecular Biology Organizationlong-term fellowship (ALTF 887-2009). M.P.W. is funded by a Well-come Trust Senior Clinical fellowship (108070/Z/15/Z). R.J.S. is sup-ported by MRC grant (MR/L008734/1). P.J.L. is supported by a Well-come Trust Principal Research Fellowship, grant (WT101835). J.S. issupported by MRC Programme grant (G0701279). J.R., L.V., andM.M. are supported by MRC as part of the core funding for the Mito-chondrial Biology Unit (MC_U105697135). L.V. is also supported byEMBO (ALFT 701-2013).

FUNDING INFORMATIONThis work, including the efforts of Michael P. Weekes, was funded byWellcome Trust (108070/Z/15/Z). This work, including the efforts of PaulLehner, was funded by Wellcome Trust (WT101835). This work, includ-ing the efforts of John Sinclair, was funded by Medical Research Council(MRC) (G0701279). This work, including the efforts of Richard JamesStanton, was funded by Medical Research Council (MRC) (MR/L008734/1). This work, including the efforts of Joanna Rorbach, Lindsey VanHaute, and Michal Minczuk, was funded by Medical Research Council(MRC) (MC_U105697135). This work, including the efforts of SharonKarniely, was funded by European Molecular Biology Organization(EMBO) (ALTF 887-2009). This work, including the efforts of LindseyVan Haute, was funded by European Molecular Biology Organization(EMBO) (ALFT 701-2013).

The funders had no role in study design, data collection and interpreta-tion, or the decision to submit the work for publication.

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